CN110283798A - A kind of purification process of gene recombinant protein Tat-hMsrA - Google Patents
A kind of purification process of gene recombinant protein Tat-hMsrA Download PDFInfo
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- CN110283798A CN110283798A CN201910616431.3A CN201910616431A CN110283798A CN 110283798 A CN110283798 A CN 110283798A CN 201910616431 A CN201910616431 A CN 201910616431A CN 110283798 A CN110283798 A CN 110283798A
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- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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Abstract
The present invention relates to the purification process of gene recombinant protein Tat-hMsrA a kind of comprising following steps: obtaining the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA；Escherichia coli bacteria liquid is centrifuged, bacterial sediment is resuspended, ultrasonication, then is centrifuged, and obtains inclusion body precipitating；Inclusion body precipitating is resuspended in cleaning solution, is sheared using high pressure homogenizer, obtains broken liquid；Wherein, cleaning solution be comprising guanidine hydrochloride, n-dodecanol sarcosine, cysteine and lysozyme phosphate buffer；Broken liquid is centrifuged, liquid is discarded supernatant, enterokinase enzyme cutting buffering liquid is added into albumen precipitation, obtains mixed solution；Renaturation solution will be added into mixed solution, renaturation obtains renaturation mixture；By renaturation mixture through column chromatographic elution, be concentrated by ultrafiltration, freeze-drying, collect target protein Tat-hMsrA to get.The purification process of gene recombinant protein Tat-hMsrA can get the higher Tat-hMsrA of activity.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of purification process of gene recombinant protein Tat-hMsrA.
Background technique
Methionine sulfoxide reductase A (MrsA) is a kind of very strong antioxidase.Cell-penetrating peptides Tat is safe and non-toxic, can
Penetrating cell, blood-brain barrier, the two combine the humanized's recombinant protein Tat-hMrsA obtained that can be expected to be used for cosmetics, health care
In product and drug.
Paper " protective effect and genetic recombination Tat- of the methionine sulfoxide reductase A to mitochondrial oxidation stress damage
It is pointed out in hMsrA pilot process research ": constructing pet32a-Tat-hMsrA plasmid using the method for genetic engineering, and from BL21
(DE3), gammi, rosseta, gold screen most suitable host strain BL21 (DE3), then carry out the isopropyl-β-D- of albumen
Thiogalactoside (IPTG) inducing expression, discovery target protein are mainly expressed in inclusion body, need to carry out the change of target protein
Property and renaturation.But protein of interest is obtained from inclusion body, it is inactive after detection.
Summary of the invention
Based on this, it is necessary to provide the purification process of gene recombinant protein Tat-hMsrA a kind of, can get active
Tat-hMsrA。
The technical scheme to solve the above technical problems is that
The present invention provides the purification process of gene recombinant protein Tat-hMsrA a kind of, includes the following steps:
S1 obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA；
The Escherichia coli bacteria liquid is centrifuged by S2, and bacterial sediment is resuspended, ultrasonication, then is centrifuged, and obtains inclusion body precipitating；
Inclusion body precipitating is resuspended in cleaning solution, is sheared using high pressure homogenizer, obtain broken liquid by S3；Wherein, institute
State cleaning solution be comprising guanidine hydrochloride, n-dodecanol sarcosine, cysteine and lysozyme phosphate buffer；
The broken liquid is centrifuged, discards supernatant liquid, enterokinase enzyme cutting buffering liquid is added into albumen precipitation, obtains mixed by S4
Close solution；
S5, will be added renaturation solution into the mixed solution, and renaturation obtains renaturation mixture；
S6 is concentrated by ultrafiltration by the renaturation mixture through column chromatographic elution, and target protein Tat- is collected in freeze-drying
HMsrA to get.
Preferably, in step s3, the cleaning solution is to include 4-8mol/L guanidine hydrochloride, 0.1-0.5mg/mL n-dodecanol
The phosphate buffer of sarcosine, 0.5-1.5mg/mL cysteine and 0.5-1.5mg/mL lysozyme, inclusion body precipitating and
The volume ratio 1:(5-20 of the cleaning solution).
It is highly preferred that the cleaning solution be comprising 5-6mol/L guanidine hydrochloride, 0.3-0.4mg/mL n-dodecanol sarcosine,
The phosphate buffer of 0.6-0.8mg/mL cysteine and 0.8-1.0mg/mL lysozyme.
Preferably, in step s 5, the renaturation solution is to include 0.5-1.5mg/mL lysozyme, the Portugal 0.5-1.5mg/L
The phosphate buffer of grape sugar and 0.5-2.0mg/mL compound amino acid.
It is highly preferred that in step s 5, the renaturation solution is to include 0.7-1.0mg/mL lysozyme, 0.8-1.2mg/L
The phosphate buffer of glucose and 1.0-1.5mg/mL compound amino acid.
Preferably, the technological parameter of the high pressure homogenizer shearing are as follows: revolving speed 500-2000r/min, time 60-
180min。
Preferably, in step s 6, the column chromatography successively includes gel exclusion chromatography and ion-exchange chromatography.
It is highly preferred that the ion-exchange chromatography uses cross-linked dextran gel column, and successively using 0,0.1mol/L,
The sodium chloride solution of 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L carry out gradient elution, collect target eluting peak.
Tat-hMsrA prepared by the purification process of gene recombinant protein Tat-hMsrA described in any of the above embodiments.
The beneficial effects of the present invention are:
Compared with prior art, the purification process of gene recombinant protein Tat-hMsrA of the invention can get active
Tat-hMsrA。
Detailed description of the invention
The SDS-PAGE figure that Fig. 1 is the target gene recombinant protein Tat-hMsrA that embodiment 3 obtains.
Specific embodiment
It is described below in conjunction with specific embodiment, the given examples are served only to explain the present invention, is not intended to limit this hair
Bright range.Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff it is identical.Term as used herein in the specification of the present invention is intended merely to describe
The purpose of specific embodiment, it is not intended that in the limitation present invention.
The purification process of the gene recombinant protein Tat-hMsrA of one embodiment, includes the following steps:
S1 obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
In this step, according to Ph.D. Dissertation " guarantor of the methionine sulfoxide reductase A to mitochondrial oxidation stress damage
Part III-genetic recombination Tat-hMsrA pilot scale route in shield effect and the research of genetic recombination Tat-hMsrA pilot process "
Following experimental method disclosed in research obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
Step S1 Escherichia coli bacteria liquid is centrifuged by S2, and bacterial sediment is resuspended, ultrasonication, then is centrifuged, and it is heavy to obtain inclusion body
It forms sediment.
Inclusion body precipitating is resuspended in cleaning solution, is sheared using high pressure homogenizer, obtain broken liquid by S3.Wherein, cleaning solution
For comprising guanidine hydrochloride, n-dodecanol sarcosine, cysteine and lysozyme phosphate buffer.
Preferably, cleaning solution is to include 4-8mol/L guanidine hydrochloride, 0.1-0.5mg/mL n-dodecanol sarcosine, 0.5-
The phosphate buffer of 1.5mg/mL cysteine and 0.5-1.5mg/mL lysozyme, the volume ratio 1 of inclusion body precipitating and cleaning solution:
(5-20)。
Broken liquid is centrifuged, discards supernatant liquid by S4, and enterokinase enzyme cutting buffering liquid is added into albumen precipitation, must mix molten
Liquid.
S5 isometric renaturation solution will be added into mixed solution, and renaturation obtains renaturation mixture.
Preferably, property solution is to include 0.5-1.5mg/mL lysozyme, 0.5-1.5mg/L glucose and 0.5-2.0mg/mL
The phosphate buffer of compound amino acid.
S6 is concentrated by ultrafiltration by renaturation mixture through column chromatographic elution, and target protein Tat-hMsrA is collected in freeze-drying,
To obtain the final product.
Active Tat- can get using the purification process of the gene recombinant protein Tat-hMsrA of present embodiment
hMsrA。
It is illustrated combined with specific embodiments below.
Embodiment 1
This implementation provides the purification process of gene recombinant protein Tat-hMsrA a kind of, includes the following steps:
S1 obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
In this step, according to Ph.D. Dissertation " guarantor of the methionine sulfoxide reductase A to mitochondrial oxidation stress damage
Part III-genetic recombination Tat-hMsrA pilot scale route in shield effect and the research of genetic recombination Tat-hMsrA pilot process "
Following experimental method disclosed in research obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
The Escherichia coli bacteria liquid of step S1 is centrifuged by S2 under the conditions of 4 DEG C, 8000g, obtains bacterial sediment, then use pH7.0
Phosphate buffer be resuspended, ultrasonication under condition of ice bath is centrifuged under the conditions of 4 DEG C, 18000g, obtains supernatant and inclusion body
Precipitating.
Inclusion body precipitating is resuspended in cleaning solution, is sheared using high pressure homogenizer by S3, under the conditions of 4 DEG C of temperature, revolving speed
1800r/min, time 80min obtain broken liquid.Wherein, cleaning solution is to include 8mol/L guanidine hydrochloride, 0.1mg/mL n-dodecanol flesh
The phosphate buffer of propylhomoserin, 1.5mg/mL cysteine and 0.5mg/mL lysozyme, the volume ratio of inclusion body precipitating and cleaning solution
1:20。
The step S3 broken liquid obtained and step the S2 supernatant obtained are merged, are centrifuged, discard in 4 DEG C, 20000g by S4
Enterokinase enzyme cutting buffering liquid is added into albumen precipitation, obtains mixed solution for supernatant.
S5 isometric renaturation solution will be added into mixed solution, and renaturation obtains renaturation mixture.Wherein, renaturation solution
For the phosphorus comprising 0.5mg/mL lysozyme, 1.5mg/L glucose and 0.5mg/mL compound amino acid (containing conventional 18 kinds of amino acid)
Acid buffer.
S6 elutes renaturation mixture through gel exclusion chromatography and ion-exchange chromatography, and ion-exchange chromatography is using friendship
Join sephadex column G-100, and successively uses 0,0.1mol/L, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L
Sodium chloride solution carry out gradient elution, collect target eluting peak (always using SDS-PAGE monitor), be concentrated by ultrafiltration, freezing is dry
It is dry, target protein Tat-hMsrA is collected, active testing is carried out.
Embodiment 2
This implementation provides the purification process of gene recombinant protein Tat-hMsrA a kind of, includes the following steps:
S1 obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
In this step, according to Ph.D. Dissertation " guarantor of the methionine sulfoxide reductase A to mitochondrial oxidation stress damage
Part III-genetic recombination Tat-hMsrA pilot scale route in shield effect and the research of genetic recombination Tat-hMsrA pilot process "
Following experimental method disclosed in research obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
The Escherichia coli bacteria liquid of step S1 is centrifuged by S2 under the conditions of 4 DEG C, 8000g, obtains bacterial sediment, then use pH7.0
Phosphate buffer be resuspended, ultrasonication under condition of ice bath is centrifuged under the conditions of 4 DEG C, 18000g, obtains supernatant and inclusion body
Precipitating.
Inclusion body precipitating is resuspended in cleaning solution, is sheared using high pressure homogenizer by S3, under the conditions of 4 DEG C of temperature, revolving speed
600r/min, time 150min obtain broken liquid.Wherein, cleaning solution is to include 4mol/L guanidine hydrochloride, 0.5mg/mL n-dodecanol flesh
The phosphate buffer of propylhomoserin, 0.5mg/mL cysteine and 1.5mg/mL lysozyme, the volume ratio of inclusion body precipitating and cleaning solution
1:5。
The step S3 broken liquid obtained and step the S2 supernatant obtained are merged, are centrifuged, discard in 4 DEG C, 20000g by S4
Enterokinase enzyme cutting buffering liquid is added into albumen precipitation, obtains mixed solution for supernatant.
S5 isometric renaturation solution will be added into mixed solution, and renaturation obtains renaturation mixture.Wherein, renaturation solution
For the phosphorus comprising 1.5mg/mL lysozyme, 0.5mg/L glucose and 2.0mg/mL compound amino acid (containing conventional 18 kinds of amino acid)
Acid buffer.
S6 elutes renaturation mixture through gel exclusion chromatography and ion-exchange chromatography, and ion-exchange chromatography is using friendship
Join sephadex column G-100, and successively uses 0,0.1mol/L, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L
Sodium chloride solution carry out gradient elution, collect target eluting peak (always using SDS-PAGE monitor), be concentrated by ultrafiltration, freezing is dry
It is dry, target protein Tat-hMsrA is collected, active testing is carried out.
Embodiment 3
This implementation provides the purification process of gene recombinant protein Tat-hMsrA a kind of, includes the following steps:
S1 obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
In this step, according to Ph.D. Dissertation " guarantor of the methionine sulfoxide reductase A to mitochondrial oxidation stress damage
Part III-genetic recombination Tat-hMsrA pilot scale route in shield effect and the research of genetic recombination Tat-hMsrA pilot process "
Following experimental method disclosed in research obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
The Escherichia coli bacteria liquid of step S1 is centrifuged by S2 under the conditions of 4 DEG C, 8000g, obtains bacterial sediment, then use pH7.0
Phosphate buffer be resuspended, ultrasonication under condition of ice bath is centrifuged under the conditions of 4 DEG C, 18000g, obtains supernatant and inclusion body
Precipitating.
Inclusion body precipitating is resuspended in cleaning solution, is sheared using high pressure homogenizer by S3, under the conditions of 4 DEG C of temperature, revolving speed
1200r/min, time 120min obtain broken liquid.Wherein, cleaning solution is to include 5.5mol/L guanidine hydrochloride, 0.3mg/mL positive 12
The phosphate buffer of alcohol sarcosine, 1.0mg/mL cysteine and 1.0mg/mL lysozyme, the body of inclusion body precipitating and cleaning solution
Product compares 1:12.
The step S3 broken liquid obtained and step the S2 supernatant obtained are merged, are centrifuged, discard in 4 DEG C, 20000g by S4
Enterokinase enzyme cutting buffering liquid is added into albumen precipitation, obtains mixed solution for supernatant.
S5 isometric renaturation solution will be added into mixed solution, and renaturation obtains renaturation mixture.Wherein, renaturation solution
For the phosphorus comprising 1.0mg/mL lysozyme, 1.0mg/L glucose and 1.3mg/mL compound amino acid (containing conventional 18 kinds of amino acid)
Acid buffer.
S6 elutes renaturation mixture through gel exclusion chromatography and ion-exchange chromatography, and ion-exchange chromatography is using friendship
Join sephadex column G-100, and successively uses 0,0.1mol/L, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L
Sodium chloride solution carry out gradient elution, collect target eluting peak (always using SDS-PAGE monitor), be concentrated by ultrafiltration, freezing is dry
It is dry, target protein Tat-hMsrA is collected, active testing is carried out.
In the present embodiment in step S6 the SDS-PAGE monitoring figure of target eluting peak as shown in Figure 1, as seen from Figure 1,
The purity of target protein Tat-hMsrA is higher.
Comparative example 1
This implementation provides the purification process of gene recombinant protein Tat-hMsrA a kind of, includes the following steps:
S1 obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
In this step, according to Ph.D. Dissertation " guarantor of the methionine sulfoxide reductase A to mitochondrial oxidation stress damage
Part III-genetic recombination Tat-hMsrA pilot scale route in shield effect and the research of genetic recombination Tat-hMsrA pilot process "
Following experimental method disclosed in research obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
The Escherichia coli bacteria liquid of step S1 is centrifuged by S2 under the conditions of 4 DEG C, 8000g, obtains bacterial sediment, then use pH7.0
Phosphate buffer be resuspended, ultrasonication under condition of ice bath is centrifuged under the conditions of 4 DEG C, 18000g, obtains supernatant and inclusion body
Precipitating.
Inclusion body precipitating is resuspended in cleaning solution, is sheared using high pressure homogenizer by S3, under the conditions of 4 DEG C of temperature, revolving speed
1200r/min, time 120min obtain broken liquid.Wherein, cleaning solution is to include half Guang ammonia of 5.5mol/L guanidine hydrochloride and 1.0mg/mL
The phosphate buffer of acid, the volume ratio 1:12 of inclusion body precipitating and cleaning solution.
The step S3 broken liquid obtained and step the S2 supernatant obtained are merged, are centrifuged, discard in 4 DEG C, 20000g by S4
Enterokinase enzyme cutting buffering liquid is added into albumen precipitation, obtains mixed solution for supernatant.
S5 isometric renaturation solution will be added into mixed solution, and renaturation obtains renaturation mixture.Wherein, renaturation solution
For the phosphate buffer comprising 1.0mg/mL lysozyme and 1.3mg/mL compound amino acid (containing conventional 18 kinds of amino acid).
S6 elutes renaturation mixture through gel exclusion chromatography and ion-exchange chromatography, and ion-exchange chromatography is using friendship
Join sephadex column G-100, and successively uses 0,0.1mol/L, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L
Sodium chloride solution carry out gradient elution, collect target eluting peak (always using SDS-PAGE monitor), be concentrated by ultrafiltration, freezing is dry
It is dry, target protein Tat-hMsrA is collected, active testing is carried out.
Comparative example 2
This implementation provides the purification process of gene recombinant protein Tat-hMsrA a kind of, includes the following steps:
S1 obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
In this step, according to Ph.D. Dissertation " guarantor of the methionine sulfoxide reductase A to mitochondrial oxidation stress damage
Part III-genetic recombination Tat-hMsrA pilot scale route in shield effect and the research of genetic recombination Tat-hMsrA pilot process "
Following experimental method disclosed in research obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA.
The Escherichia coli bacteria liquid of step S1 is centrifuged by S2 under the conditions of 4 DEG C, 8000g, obtains bacterial sediment, then use pH7.0
Phosphate buffer be resuspended, ultrasonication under condition of ice bath is centrifuged under the conditions of 4 DEG C, 18000g, obtains supernatant and inclusion body
Precipitating.
Inclusion body precipitating is resuspended in cleaning solution, is sheared using high pressure homogenizer by S3, under the conditions of 4 DEG C of temperature, revolving speed
1200r/min, time 120min obtain broken liquid.Wherein, cleaning solution is to include 1.0mg/mL cysteine and 1.0mg/mL bacteriolyze
The phosphate buffer of enzyme, the volume ratio 1:12 of inclusion body precipitating and cleaning solution.
The step S3 broken liquid obtained and step the S2 supernatant obtained are merged, are centrifuged, discard in 4 DEG C, 20000g by S4
Enterokinase enzyme cutting buffering liquid is added into albumen precipitation, obtains mixed solution for supernatant.
S5 isometric renaturation solution will be added into mixed solution, and renaturation obtains renaturation mixture.Wherein, renaturation solution
For the phosphate buffer comprising 1.0mg/mL lysozyme.
S6 elutes renaturation mixture through gel exclusion chromatography and ion-exchange chromatography, and ion-exchange chromatography is using friendship
Join sephadex column G-100, and successively uses 0,0.1mol/L, 0.2mol/L, 0.4mol/L, 0.6mol/L, 0.8mol/L
Sodium chloride solution carry out gradient elution, collect target eluting peak (always using SDS-PAGE monitor), be concentrated by ultrafiltration, freezing is dry
It is dry, target protein Tat-hMsrA is collected, active testing is carried out.
Active testing, egg are carried out to the target protein Tat-hMsrA that embodiment 1 to 3, comparative example 1 and 2 prepare respectively
Method described in patent of the white determination of activity using CN102094068A, statistical result see the table below 1:
The protein active summary sheet of 1 embodiment 1 to 3 of table and comparative example 1 and 2
By table 1, it is apparent that compared with comparative example 1 and 2, using the gene recombinant protein Tat- of embodiment 1 to 3
The purification process of hMsrA can integrally obtain the higher recombination albumen Tat-hMsrA of activity, up to electrophoresis pure 90% or more.
Each technical characteristic of above embodiments can be combined arbitrarily, for simplicity of description, not to above-described embodiment
In each technical characteristic it is all possible combination be all described, as long as however, the combination of these technical characteristics be not present lance
Shield all should be considered as described in this specification.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, it is all in spirit of the invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of purification process of gene recombinant protein Tat-hMsrA, which comprises the steps of:
S1 obtains the Escherichia coli bacteria liquid of expressed fusion protein Trx-Tat-hMsrA；
The Escherichia coli bacteria liquid is centrifuged by S2, and bacterial sediment is resuspended, ultrasonication, then is centrifuged, and obtains inclusion body precipitating；
Inclusion body precipitating is resuspended in cleaning solution, is sheared using high pressure homogenizer, obtain broken liquid by S3；Wherein, described to wash
Wash liquid be comprising guanidine hydrochloride, n-dodecanol sarcosine, cysteine and lysozyme phosphate buffer；
The broken liquid is centrifuged, discards supernatant liquid, enterokinase enzyme cutting buffering liquid is added into albumen precipitation, must mix molten by S4
Liquid；
S5, will be added renaturation solution into the mixed solution, and renaturation obtains renaturation mixture；
S6 is concentrated by ultrafiltration by the renaturation mixture through column chromatographic elution, and target protein Tat-hMsrA is collected in freeze-drying,
To obtain the final product.
2. the purification process of gene recombinant protein Tat-hMsrA according to claim 1, which is characterized in that in step S3
In, the cleaning solution is to include 4-8mol/L guanidine hydrochloride, 0.1-0.5mg/mL n-dodecanol sarcosine, half Guang of 0.5-1.5mg/mL
The phosphate buffer of propylhomoserin and 0.5-1.5mg/mL lysozyme, the volume ratio 1:(5- of the inclusion body precipitating and the cleaning solution
20)。
3. the purification process of gene recombinant protein Tat-hMsrA according to claim 2, which is characterized in that the washing
Liquid is to include 5-6mol/L guanidine hydrochloride, 0.3-0.4mg/mL n-dodecanol sarcosine, 0.6-0.8mg/mL cysteine and 0.8-
The phosphate buffer of 1.0mg/mL lysozyme.
4. the purification process of gene recombinant protein Tat-hMsrA according to claim 2, which is characterized in that in step S5
In, the renaturation solution is to include 0.5-1.5mg/mL lysozyme, 0.5-1.5mg/L glucose and the compound ammonia of 0.5-2.0mg/mL
The phosphate buffer of base acid.
5. the purification process of gene recombinant protein Tat-hMsrA according to claim 4, which is characterized in that in step S5
In, the renaturation solution is to include 0.7-1.0mg/mL lysozyme, 0.8-1.2mg/L glucose and the compound ammonia of 1.0-1.5mg/mL
The phosphate buffer of base acid.
6. the purification process of gene recombinant protein Tat-hMsrA according to claim 1, which is characterized in that the high pressure
The technological parameter of homogenizer shearing are as follows: revolving speed 500-200r/min, time 60-180min.
7. the purification process of gene recombinant protein Tat-hMsrA according to any one of claims 1 to 6, which is characterized in that
In step s 6, the column chromatography successively includes gel exclusion chromatography and ion-exchange chromatography.
8. the purification process of gene recombinant protein Tat-hMsrA according to claim 7, which is characterized in that the ion
Exchange column chromatography uses cross-linked dextran gel column, and successively uses 0,0.1mol/L, 0.2mol/L, 0.4mol/L, 0.6mol/
L, the sodium chloride solution of 0.8mol/L carries out gradient elution, collects target eluting peak.
9. Tat- prepared by the purification process of the described in any item gene recombinant protein Tat-hMsrA of claim 1 to 8
hMsrA。
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112522223A (en) * | 2021-02-18 | 2021-03-19 | 天津科技大学 | Genetically engineered bacterium for producing L-sarcosine and construction method and application thereof |
| CN113088499A (en) * | 2021-03-11 | 2021-07-09 | 嘉兴玖肽生物技术有限公司 | High-purity purification method of gene recombinant protein Tat-hMsrA |
| US11479795B2 (en) | 2021-02-18 | 2022-10-25 | Tianjin University Of Science And Technology | Genetically engineered bacterium for sarcosine production as well as construction method and application |
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| CN112522223A (en) * | 2021-02-18 | 2021-03-19 | 天津科技大学 | Genetically engineered bacterium for producing L-sarcosine and construction method and application thereof |
| CN112522223B (en) * | 2021-02-18 | 2021-05-25 | 天津科技大学 | Genetically engineered bacterium for producing L-sarcosine and construction method and application thereof |
| US11479795B2 (en) | 2021-02-18 | 2022-10-25 | Tianjin University Of Science And Technology | Genetically engineered bacterium for sarcosine production as well as construction method and application |
| CN113088499A (en) * | 2021-03-11 | 2021-07-09 | 嘉兴玖肽生物技术有限公司 | High-purity purification method of gene recombinant protein Tat-hMsrA |
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